Proceeding8 of the National Academny of SciencesVol. 66, No. 3, pp. 738-744, July 1970

Subunit Structure of Human Fibrinogen, Soluble Fibrin,and Cross-Linked Insoluble Fibrin*

Patrick A. McKee, Patrick Mattock, and Robert L. HilltDEPARTMENTS OF BIOCHEMISTRY AND MEDICINE, DUKE UNIVERSITY MEDICAL CENTER, DURHAM,

NORTH CAROLINA

Communicated by Philip Handler, April 8, 1970

Abstract. The three unique polypeptide chains of human fibrinogen differsignificantly in molecular weight. Cross-linkage of fibrin by fibrin-stabilizingfactor results in the rapid formation of cross-links between y-chains and a slowerformation of cross-links between a-chains. p-Chains are not involved directlyin the cross-linking of fibrin. Reduced, cross-linked fibrin contains uncross-linked i-chains, dimers of y-chain, and higher polymers of a-chain. Althoughit is uncertain whether the y-,y dimers are formed by chains in different moleculesof fibrin, the polymers of a-chain in fibrin can only be accounted for by cross-linkage of a-chains in different molecules. The nature of cross-linkage amongthe subunits in fibrin can account well for the three-dimensional, covalent struc-ture of cross-linked, insoluble fibrin.

Human fibrinogen, which has a molecular weight of approximately 330,000,contains three pairs of different polypeptide subunits (a, Al, y) and may bedesignated by the polypeptide chain formula a38272.1-4 The three chains arecombined covalently through disulfide bonds and differ in amino acid sequenceand molecular weight. It has been estimated that the molecular weights of thea-, (3-, and 7-chains are 63,500, 56,000, and 47,000, respectively.4 Fibrin isformed when thrombin acts on fibrinogen and cleaves specific peptide bonds in thea- and ,8-chains, thereby releasing low molecular weight peptides. Thrombinaction alone produces fibrin which is less soluble than fibrinogen but is soluble atlow pH or in concentrated solutions of urea or guanidine hydrochloride. Suchfibrin is termed noncross-linked, soluble fibrin. Soluble fibrin is converted tocross-linked, insoluble fibrin by a transpeptidase, termed fibrin-stabilizing factor(FSF), which catalyzes the formation of new amide bonds between e-aminogroups of lysine and carboxamido groups of glutamine in the subunit chains.5Thus, cross-linked, insoluble fibrin, after reduction of its disulfide bonds, con-tains higher molecular weight subunits than fibrinogen. Chen and Doolittle6concluded that reduced, cross-linked bovine fibrin contains y-7y as well as a--ydimers, whereas Lorand and co-workers found mainly a--y dimers.7 8 Similarly,Takagi and Iwanaga9 suggest that two to four 7-chains are cross-linked in in-soluble fibrin.We wish to report here the results of studies on the subunit structure of human

fibrinogen and cross-linked, insoluble, human fibrin. During the course of the738

VOL. 66, 1970 BIOCHEMISTRY: McKEE, MATTOCK, AND HILL 739

formation of insoluble fibrin we have found a rapid introduction of cross-linksbetween y-chains which lead to the formation of 'y-"y dimers in reduced fibrin anda much slower formation of a-a cross-links which lead to a mixture of highermolecular weight polymers of a-chain. There was no indication of a--y cross-linked subunits.

Experimental Procedure. Fibrinogen and fibrin preparations: Human fibrin-ogen (98% clottable) was prepared as described earlier10 and stored lyophilized at -20oC.Noncross-linked, soluble fibrin is defined as fibrin which was soluble within 15 min in 10 Murea at 250 C and was prepared as follows. Bovine thrombin (0.05 ml, containing 25 NIHunits) was added to a solution of fibrinogen (3.2 ml containing 2 mg of fibrinogen) dis-solved in 0.031 M sodium phosphate buffer, pH 6.4, containing 0.014 M calcium chloride.After 2 hr at 250C, the fibrin clot was removed, suspended for 5 min in 100 ml of0.15 M sodium chloride, and blotted with filter paper to remove excess liquid. Cross-linked, insoluble, human fibrin, which is defined as fibrin insoluble after 100 hr in a solutionof 10 M urea at 250C, was prepared as follows. A solution of fibrinogen was treated withthrombin exactly as described above for preparation of noncross-linked, soluble fibrin, and0.1 ml of fresh plasma was added as a source of FSF. After the desired extent of reaction(1 min to 24 hr), the fibrin was collected as described above. Alternatively, cross-linked,insoluble fibrin was prepared from human plasma by substitution of plasma for thefibrinogen solution. Reduced fibrinogen and reduced fibrin were prepared by incubating1-2 fug of fibrinogen, noncross-linked fibrin, or cross-linked fibrin in 1 ml of 0.05M sodiumphosphate buffer, pH 7, containing 1% sodium dodecyl sulfate, 0.1 M f-mercaptoethanol,and 4 M urea. Reduction was allowed to proceed at 370C for 2 hr with fibrinogen and24 hr with the fibrin preparations. Aliquots of the reduced proteins were subjecteddirectly to gel electrophoresis without further treatment. S-sulfofibrinogen and the S-sulfo-a-, #-, and 7-chains were prepared as described earlier.4Reagents: Bovine thrombin (Parke-Davis) was dissolved in 0.075 M sodium

chloride containing 50% glycerol to give a concentration of 500 NIH units/ml and storedat -200C. Human plasma was prepared from fresh whole blood collected with one partanticoagulant (3.5% sodium citrate) to nine parts whole blood. All other reagents wereanalytical grade unless stated otherwise.

Gel electrophoresis in sodium dodecyl sulfate: The method employed wasessentially identical to those described earlier."" 2 Throughout this study, 5% acryl-amide gels were employed in 15 X 0.6-cm glass columns. Between 0.01 and 0.05 mg ofprotein was applied per gel. Electrophoresis was performed at 20-250C at a constantcurrent of 9 mA per tube for 7 hr, by which time the marker dye (0.05% bromphenol bluein water) had migrated to within 1 cm of the bottom of the gel. The gels were stained in asolution of Coomassie brilliant blue for 12 hr and destained for 24 hr in a solution con-taining 14% acetic acid and 7% methanol. The molecular weights of the subunits offibrinogen and fibrin were determined from their mobilities and the mobilities of proteinsof known molecular weight as described earlier."1',2Amino acid composition: The amino acid composition of the subunit polypep-

tides from fibrinogen or fibrin was estimated by analysis of acid hydrolysates of slices ofelectrophoresis gels containing the appropriate stained protein bands. After electro-phoresis of 0.1 mg of a fibrinogen or fibrin preparation in sodium dodecyl sulfate gels asdescribed above, the stained bands corresponding to a single protein species were cutfrom the gel with a razor blade. Five bands from separate gels were combined, washedtwice by suspension in 5-10 ml of 6 N HCl, and then hydrolyzed at 1100C for 24 hr underreduced pressure in 2 ml of 6 N HCl containing 0.05 ml of mercaptoacetic acid. The in-soluble residue remaining after hydrolysis was removed after cooling the hydrolysate tonear its freezing point. The resulting hydrolysate was analyzed on a Beckman 120Bamino acid analyzer equipped with high-sensitivity cuvettes.Experimental Results. Gel electrophoretic analysis of fibrinogen: When

reduced and unreduced fibrinogen were submitted to electrophoresis on poly-

740 BIOCHEMISTRY: McKEE, MATTOCK, AND HILL PROC. N. A. S.

a-polymer----- ---->400-- ----- ------- 330,000~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~3300Whele Fibrinogn--------

Idi ;..

-monmer-- ..... .- O-T-- lAhn:

n-monomera-monomer--- ...ad6 0

Whole Reduced Solube Partinr y I tk MWFibrimqen. Fibrineen Fibrin $kD eble jbr

FIG. 1.-Gel electrophoresis of fibrinogen and fibrin insodium dodecylsulfate.

acrylamide gels in sodium dodecyl sulfate, patterns such as those shown inFigure 1 were obtained. Unreduced fibrinogen gave a single species whichmigrated as a protein of molecular weight 330,000, a value in good agreementwith the observed molecular weight of fibrinogen determined by other means.3'4In contrast, reduced fibrinogen gave three species which corresponded to pro-teins of molecular weight 73,000, 60,000, and 53,000. Figure 2 shows that eachof the three bands in reduced fibrinogen corresponds to one of the three uniquechains obtained on chromatography of S-sulfofibrinogen on carboxymethyl-cellulose. y-Chain, which is the first peak eluted from the chromatographiccolumn, migrates with a molecular weight of 53,000, a value in close accord withthe molecular weight of 47,000 estimated earlier by ultracentrifugation.4 Thesecond peak from the column contains S-sulfo-,B-chain, which migrates as thespecies of 60,000 molecular weight, a value also in good agreement with the previ-

1.0 ----- - - -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~001----

E~~~~~~~~0.8 ~~~~~~~~~~~~~~~034

1.07

O100200300 400 500 600 700 800 6000

0.5

0 0.4

~0.7 -00.2----- -~~~~~~~~~~~~~~~~~.20

0.1-~~~~~~~~~~~~~~/3 a~~~~~~~~.

0 100 200 300 400 500 600 700 800ELUTION VOLUME (ml)

FIG. 2.-(A) Chromatographic separation of the a-, fl-, and -y-chains of sulfofibrinogen.(B) Gel electrophoresis of the S-sulfo-a-, /3-, and 7-chains of fibrinogen in sodium dodecylsulfate.

VOL. 66, 1970 BIOCHEMISTRY: MCKEE, MATTOCK, AND HILL 741

ously reported molecular weight of 56,000 for this chain.4 The third peak fromthe column, which is S-sulfo-a-chain, migrated as a species of 73,000 molecularweight, a value somewhat higher than a weight of 63,500 reported earlier.4However, the a-chain peak also contained higher molecular weight materialwhose migration on the gels suggested that it corresponded to unreduced fibrin-ogen. This suggestion was confirmed when it was found to give rise to bandscorresponding to f3- and y-chains, in-addition to a-chain, after reduction and gelelectrophoretic analysis. Finally, it should be emphasized that a-chain fromseveral different samples of fibrinogen was somewhat heterogeneous in size.Although it is difficult to see in the figures, two and sometimes three bands,ranging in molecular weight from 68,000 to 76,000, were observed on gel elec-trophoresis.Gel electrophoresis of soluble and partially soluble fibrin: Figure 1 also

shows the behavior of reduced, soluble fibrin on dodecyl sulfate gels. It is seenthat the patterns obtained were identical to those given by reduced fibrinogenexcept that the a-chain migrated as a species of molecular weight 68,000 andwas not as heterogeneous as seen in fibrinogen.

Partially soluble' fibrin which was formed from fibrinogen after the action ofthrombin and FSF for 1 min gave the gel patterns indicated in Figure 1 afterreduction. It is clear that the a- and #-chains migrate as found in fibrinogen andsoluble fibrin, whereas the 7-chains have almost completely disappeared. Fur-thermore, a species of molecular weight 105,000 appeared. These data suggestthat an early event in the formation of cross-linked fibrin is the formation ofcross-links between two 7-chains.Gel electrophoresis of insoluble, cross-linked fibrin: Figure 1 also shows the

gel electrophoretic behavior of insoluble, cross-linked fibrin which was formedfrom fibrinogen after treatment with thrombin and FSF for 2 hr. It can be seenthat 7-chains as well as a-chains are absent in these preparations, although theA-chains migrate identically with those in reduced fibrinogen or reduced solublefibrin. In addition to a major species which migrates with a molecular weight of105,000, the reduced cross-linked insoluble fibrin contains a component whichdoes not penetrate the gels and must correspond to a protein species of a molec-ular weight greater than 400,000, the exclusion limit of these gels. Thus, thesedata suggest that on prolonged action of FSF, the a-chains of fibrin are cross-linked extensively to give high molecular weight polymers.

Extent of cross-lnking in the fibrin chain with time: Figure 3 shows the gelelectrophoretic patterns of fibrin as a function of time of cross-linking with FSF.The formation of y-dimers is complete within 5 min of the onset of clotformation, although the formation of a-chain polymers is complete only after 90min. It is noteworthy that the extent of cross-linking in fibrin as judged by thegel pattern is reflected by the solubility of the fibrin in 10 M urea: the moreextensive the cross-links in a-chain, the greater the insolubility of the fibrin.Clots isolated from whole plasma gave identical results. Formation of the y--yand the -a-a- cross-links was accelerated by calcium, but both types of subunitswere formed in the presence of excess EDTA, in agreement with a recent re-port. 13

742 BIOCHEMISTRY: McKEE, MATTOCK, AND HILL PROC. N. A. S.

a-pc.Iymer..

FIG. 3.-Gel electrophoresis offibrin as a function of time of

.dAner * * * cross-linking by fibrin-stabilizingfactor. Fibrinogen was reactedSrnerthmer £

* * | | * with thrombin and the aboved mono1mer factor for the intervals indicated

as described in the text, and theresulting fibrin was reduced andanalyzed on the gels.

0 A.QTION F CLOITTC 5 53I 60 90 120

The amino acid composition of the subunit chains of fibrinogen and fibrin:Table 1 lists the amino acid compositions of each of the unique polypeptidechains of fibrinogen and fibrin. It is noteworthy that the molecular weight sub-unit ('y-'y dimer), which is formed early in the cross-linking of fibrin, has anamino acid composition that is indistinguishable, within experimental error,from the composition of y-chains of fibrinogen. The compositions of the 38-chains, which do not appear to participate in the cross-linking of fibrin, are alsoessentially identical to those for the 3-chains in cross-linked fibrin and fibrinogen.The amino acid compositions of the i3- and y-chains are also in good agreementwith compositions reported earlier.4 The composition of the a-chains of fibrin-ogen is also similar to the compositions of the higher molecular weight (>400,000)species found in extensively cross-linked fibrin. The composition of a-chain

TABLE 1. The amino acid composition of the polypeptide chains offibrinogen and fibrin*Fibrinogen Fibrin

---(residues per molecule) -4(residues per molecule)--Amino acid a-Chain f-Chain y-Chain a-Polymer f-Chain 'y-Dimer

Lysine 33.2 34.1 35.4 42.5 35.6 37.1Histidine 15.5 8.2 9.0 15.8 7.9 9.6Arginine 32.9 23.6 10.3 37.6 26.3 11.3Aspartic acid 74.7 58.1 63.9 64.0 59.2 56.6Threonine 53.9 28.1 33.1 54.6 26.8 36.1Serine 87.4 34.8 27.0 69.2 33.4 29.8Glutamic acid 75.1 67.8 55.4 70.4 61.0 54.6Proline 49.9 33.4 20.4 44.9 36.3 24.3Glycine 79.5 52.4 42.5 73.6 47.0 42.5Alanine 26.3 27.8 30.7 28.7 27.5 30.3Valine 27.6 24.5 16.0 30.7 26.1 16.4Isoleucine 15.8 15.6 23.5 21.1 16.5 22.3Leucine 36.1 33.9 32.5 37.2 34.3 30.7Tyrosine 11.7 21.9 22.5 15.4 22.1 21.2Phenylalanine 17.9 11.6 19.8 22.2 12.6 20.8

* The compositions were calculated on the basis of the following molecular weights: a-chain anda-polymer, 73,000; f-chain, 60,000; ey-chain and 7-dimer, 53,000. The tryptophan, methionine,and half-cystine contents of the chains were not estimated. No corrections were made for destruc-tion of amino acids during hydrolysis. In these calculations, it was assumed that the tryptophan,methionine, and half-cystine contents were the same as reported earlier' and the carbohydrate con-tents of the a-, fi-, and y-chains were 1, 3, and 3%, respectively.14

VOL. 66, 1970 BIOCHEMISTRY: MCKEE, MATTOCK, AND HILL 743

differs somewhat from published values,4 but the earlier values are probably inerror because of significant contamination with fibrinogen, as shown in Figure 2.

Discussion. The studies reported here confirm our earlier report4 that thethree subunit polypeptide chains of human fibrinogen differ in molecular weight.Although it has been suggested that the three subunits are of approximately thesame molecular weight since a mixture of the chains did not show gross ultra-centrifugal heterogeneity,3 the present studies confirm the view that the molec-ular weights of the chains differ significantly. By gel electrophoresis in sodiumdodecyl sulfate, the weights of the ,3- and y-chains appear to be 60,000 and53,000, respectively, in good agreement with the values of 56,000 and 47,000reported earlier. However, the molecular weight of the a-chain as estimated bygel electrophoresis is about 73,000, in contrast to a value of 63,500 obtained byultracentrifugation. It would appear that this discrepancy, which is not withinthe experimental error of the methods, may have resulted from contamination ofa-chains with intact fibrinogen, as shown in Figure 2. The combination of S-sulfo-a-chains with fibrinogen may not have been detected heretofore becauseof the similar mobilities of the two proteins in the electrophoretic systems em-ployed to judge the purity of the chains.2"4 Similarly, the contamination of a-chains with fibrinogen would not be noted in the ultracentrifuge in mercapto-ethanol and guanidine hydrochloride, the conditions used in the earlier analyses.Although the molecular weight of the a-chains appears to be about 73,000 by gelelectrophoresis, this method also suggests that the a-chains may be hetero-geneous, with molecular weights ranging from 68,000 to 76,000. Although thisheterogeneity remains unexplained, it is possible that a-chains vary in carbo-hydrate content or are partially degraded in vivo.The present studies also define the types of covalent interactions which occur

among the subunit polypeptides on cross-linking of the fibrin chains. From thedata in Figures 1 and 3 and the amino acid compositions in Table 1, it appearsthat during fibrin formation in the presence of FSF, the a- and y-chains, but notthe 8-chains, are cross-linked. There is a rapid formation of cross-links solelybetween y-chains such that in reduced fibrin only -y-'y dimers of 105,000 molec-ular weight are observed. In contrast, there is a much slower formation ofcovalent cross-links among a-chains. However, the cross-linkage among a-chains is extensive and higher molecular weight polymers of a-chain are formedinvolving at least six and perhaps more chains. Although it is impossible tojudge from the present studies whether cross-linkage between 7-chains is anintra- or intermolecular molecular reaction, it is evident that higher molecularweight a-chains could only be formed by cross-linkage between a-chains indifferent molecules. Thus, the extensive cross-linkage among fibrin molecules onformation of insoluble clots must be in large part the result of the formation ofcross-links between a-chains, without involvement of 1-chains and perhaps 7-chains. This view is consistent with the observation (Fig. 3) that as extensivecross-linkage among a-chains occurs, the resulting fibrin is rendered more in-soluble in concentrated solutions of urea.Our results on the cross-linkage of the fibrin subunit polypeptides differ some-

what from those reported recently by others. Chen and Doolittle6 suggested

744 BIOCHEMISTRY: McKEE, MATTOCK, AND HILL PROC. N. A. S.

that cross.linking of fibrin proceeds by formation of y-7y as well as a-'y cross-links. According to their scheme, equal proportions of y-y dimers and a--ydimers would be found in reduced, cross-linked fibrin and cross-linking betweenboth types of chains would occur intermolecularly. Lorand et al.7'8 also sug-gested that cross-linkage of fibrin results mainly in the formation of cross-linksbetween a- and y-chains in different fibrin molecules. In contrast, Takagi andIwanaga9 indicated that only y-chains are involved in the cross-linkage reactionsand suggested that y-y dimers as well as trimers and tetramers may be found inreduced fibrin.Although the results presented here differ somewhat from earlier schemes for

the cross-linkage of fibrin, they are in general agreement with earlier observa-tions. Waugh and Livingstonell concluded from analysis of fibrin formation bymeans of formal titrations that two reactions, which proceed at different rates,are involved. Furthermore, Lorand et al. noted that 'vchains contain an aminoacid side chain which is involved in cross-linkage that is more reactive than thosefound in other chains, presumably the a-chains.

Results essentially identical to those reported here were found with bovinefibrinogen and fibrin.

* This work was supported by research grants from the National Heart Institute, NationalInstitutes of Health (HE06400 and HE06233), and the National Science Foundation (GB-12676).

t Requests for reprints may be addressed to Dr. R. L. Hill, Departments of Biochemistryand Medicine, Duke University Medical Center, Durham, N.C. 27706.

1 Blomback, H., and I. Yamashina, Ark. Kemi, 12, 299 (1958).'Henschen, A., Ark. Kemi, 22, 375 (1964).Johnson, P., and E. Mihalyi, Biochim. Biophys. Acta, 102, 467 (1965).

4 McKee, P. A., L. A. Rogers, E. Marler, and R. L. Hill, Arch. Biochem. Biophys., 116, 271(1966).

6 Loewy, A. G., in Fibrinogen, ed. K. Laki (New York: Marcel Dekker, Inc., 1968), pp.185-.224.

X Chen, R., and R. F. Doolittle, these PROCSEDINGS, 63, 420 (1969).7 Lorand, L., D. Chenoweth, and R. A. Domanik, Biochem. Biophys. Res. Commun., 37, 219

(1969).8 Lorand, L., and D. Chenoweth, these PROCEEDINGS, 63, 1247 (1969).9 Takagi, T., and S. Iwanaga, Biochem. Biophys. Res. Commun., 38, 129 (1970).

10 Blomback, B., and M. Blomback, Ark. Kemi, 10, 415 (1956).1 Weber, K., and M. Osborn, J. Biol. Chem., 244, 4406 (1969).12 Dunker, A. K., and R. R. Rueckert, J. Biol. Chem., 244, 5074 (1969).13 Tsynkalovskaya, S. N., T. V. Varetskaya, T. F. Galanova, and V. A. Belitser, Mol. Biol.

(Enl.), 3, 422 (1969).14 Mills, D. A., and D. C. Triantaphyllopoulos, Arch. Biochem. Biophys., 135, 28 (1969).15 Waugh, D. F., and B. J. Livingstone, Science, 113, 121 (1951).